Con
Authors: Börje Haraldsson, William M. Deen
Journal: Journal of the American Society of Nephrology (2008)

Glomerulonephritis is characterized by proteinuria, hematuria, elevated BP, and morphological changes in glomeruli.1,2 It is now known that alterations of certain podocyte-specific proteins, such as nephrin, podocin, or α-actinin-4, can cause hereditary nephrotic syndromes.3 Also, there is overwhelming functional evidence for the glomerular barrier being highly size- and charge-selective, with data obtained by micropuncture,4 kidney perfusion,5,6 tissue uptake,7 and in patients with defects in the tubular reabsorption of proteins.8,9 There is, however, an alternative view (the Pro argument), according to which the proteinuria that occurs in renal disorders results entirely from defects in the tubular system with glomerular barrier function remaining normal.10 According to this view, large amounts of albumin are filtered even under normal circumstances but are reabsorbed intact by the tubules. This albumin retrieval hypothesis seems to be incompatible with most studies of podocyte biology and glomerular function.2 The albumin retrieval hypothesis10 rests on the following assertions, which will be discussed in turn: (a) there is no evidence of glomerular charge-selectivity, because studies using dextran sulfate are invalid10 and protein data indicate no charge barrier;11 (b) albumin is filtered in large quantities across the glomerular wall;10 (c) albumin is reabsorbed in intact form10 in the proximal tubules; (d) proteins are degraded and excreted in urine as small peptides,12 and; (e) special techniques are required to detect the true amounts of protein in urine.10 Of course, these assertions are interrelated. If charge-selectivity is absent from the glomerular barrier, it is much more difficult then to exclude albumin from the filtrate and much more necessary for the tubules to somehow retrieve it. If protein degradation or flaws in analytical methods cause a systematic underestimation of urinary albumin and/or total protein excretion, then once again the glomerular barrier may be less effective than widely thought. Regarding assertion (a), dextran sulfate has been shown to bind to plasma proteins, but after corrections for this there still is a significant negative charge barrier.1 The cellular uptake and binding of dextran sulfate by glomerular cells is rapidly saturable10 and therefore would not influence steady-state clearance data.1 Protein data also suggest the glomerular barrier is indeed charge-selective.5,7 This is true even for data from the Comper laboratory.11 Thus, the fractional clearance of neutral horseradish peroxidase (nHRP) was 2.4 times higher than that of anionic horseradish peroxidase (aHRP) under control conditions, a ratio identical to that reported by Sörensson et al.5 Adding 150 mM lysine or 10 mM NH4Cl to the perfusate elevated the fractional clearance of nHRP by 70% and of aHRP by 170%, giving a residual charge-selectivity ratio of 1.6.11 The original data of Osicka et al. for albumin also suggest charge-selectivity; in the isolated perfused kidney at 37°C, the fractional clearance was 0.0075 for native albumin and 0.0330 for neutral albumin.11 Similar levels of clearance were reported for neutral albumin by Bertolatus and Hunsicker using a tissue-uptake technique,7 but their clearance ratio was 43. The fact that tubular cell inhibitors raise the clearances for both the neutral and anionic proteins suggests they are toxic to the glomerular barrier. Indeed, such toxic effects were found by Ohlson et al.6 Thus, the use of lysine or NH4Cl is likely to underestimate the charge-selectivity of the glomerular barrier. Concerning assertion (b), which is that large amounts of albumin cross the glomerular barrier, the most recent estimate of the glomerular sieving coefficient of albumin from the Comper laboratory is 0.034.10 If this value (from rats) is applied to humans, the reabsorptive burden that it implies for the proximal tubules is enormous. For example, with 180 L of glomerular filtrate produced per day in man and a serum albumin level of 40 g/L, the filtered load of albumin would be 180 × 40 × 0.034 = 240 g/d. Unless albumin concentrations measured by micropuncture in proximal tubules4 are orders of magnitude too low, almost all of this filtered load would need to be reabsorbed in the first millimeter or so of tubule. An analysis based on engineering concepts that have been applied for many years to the design of tubular reactors suggests that such a high rate of albumin reabsorption would be impossible, even if every albumin molecule that reaches an epithelial cell were internalized immediately.13 In contrast, many studies suggest that only small amounts of protein normally cross the glomerular barrier, which seems to have a sieving coefficient for albumin two to three orders of magnitude <0.034.2,4,5,7,8 There are ample experimental data to rebut assertion (c), which is that albumin is reabsorbed in intact form. Forty years ago Maunsbach found no evidence for any tubular uptake of intact autologous rat albumin.14 Today, we know that albumin is taken up by the megalin-cubulin complex,15 which is controlled by a specific chloride channel, ClC-5.16 There is an obligatory coupling between uptake and degradation in lysosomes that is incompatible with the notion of reabsorption of intact albumin.15 Thus, the suggestion of Russo et al.10 that the megalin-cubulin complex could be responsible for “albumin retrieval” lacks support.9,16,17 Assertion (d), the notion that there is substantial protein degradation in the urine that might cause protein excretion to be underestimated, also lacks strong support. The application of state-of-the-art methods for protein analysis to samples from patients with Fanconi syndrome and normal controls did not reveal significant fragmentation of albumin in the urine.9 Results based on the same proteomics methodology cast doubt also on assertion (e), which is that special techniques based on the Biuret reagent and high-performance liquid chromatography are needed to detect albumin fragments in urine.10 When the sensitivity and specificity of these methods were tested, serious flaws were found. Thus, Norden et al.9 selected 14 nonpeptide components from urine samples and erroneously obtained protein concentrations of 0.4 g/L using the Biuret reagent or the high-performance liquid chromatography technique. There are data with radiolabeled albumin that have been interpreted as proof of the albumin retrieval hypothesis.12 However, the authors neglected to take into account the effects of small quantities of free tracer. Thus, even if 99.9% of the radioactivity was bound to albumin, if the unbound tracer was filtered freely and the albumin sieving coefficient was 0.0001, then there would still be 10 times more free tracer than radiolabeled albumin in the urine (99.9% × 0.0001)/(0.1% × 1) = 10. The results that have been taken as evidence of massive degradation of protein in urine have come from 3H counting of urine samples fractionated by gel filtration,12 using columns that cannot distinguish free tracer from small peptide fragments. The lack of evidence for protein degradation from the proteomics analysis, together with the sample calculation above, strongly suggests the data on urine radioactivity have been misinterpreted. This apparent misinterpretation has propagated through studies of various glomerular diseases,10,12 leading to the mistaken conclusion that barrier function is unaltered. How much albumin is filtered normally? The aforementioned study of Fanconi syndrome9 provides what is perhaps the best estimate for the albumin sieving coefficient in normal humans, namely, 0.00008. With the water filtration rate and serum concentration used earlier, this gives a daily filtered load of 180 × 40 × 0.00008 = 0.6 g/d. Even if inhibition of protein reabsorption in the Fanconi subjects were incomplete (say, only 50%), the estimate of normal albumin filtration would still be only approximately 1 g/d. Thus, we conclude that, whereas tubular defects may certainly contribute to proteinuria, defects in the glomerular barrier are almost certainly required for proteinuria to reach nephrotic levels (>3.5 g/d). In summary, there is massive documentation to support the concept of a glomerular barrier that is normally very size and charge-selective, but which can be compromised either by disease or by specific genetic variations. That is, only modest amounts of albumin normally cross the barrier, but the amounts can increase greatly if there are barrier defects. There also is firm support for the concept that filtered albumin binds to the megalin-cubulin complex of proximal tubule cells and is internalized, degraded, and released to blood as amino acids, rather than as intact albumin. No molecular mechanism has been identified for reabsorbing hundreds of grams per day of albumin. To adhere to the idea that nephrotic range proteinuria results mainly from tubular defects is to deny the existence of numerous physiologic, morphologic, and molecular biologic findings by many laboratories. DISCLOSURES None. The authors are grateful for financial support of this work from the Swedish Research Council 9898.